The present invention relates to surfaces, such as those of multiwell or microtiter plates, coated with proteins useful as capturing devices for antigens, antibodies and the like and, more particularly, to surfaces coated with polymerized streptavidin, avidin or NeutrAvidin(trademark) deglycosylated avidin.
Proteins coated on various solid surfaces are used as capturing devices for antigens, antibodies, ligands, receptors, oligonuceotides, and the like target molecules. In some cases the capturing molecules are bound directly to the surface and in other cases they are bound indirectly using, for instance, techniques based on avidin-biotin chemistry.
Solid surfaces to which the proteins are bound can be polystyrene, polypropylene, or membranes. The surfaces typically are located on the inside of tubes or the wells of plates such as microwell or microtiter plates, the bottoms of filter plates or the outside surfaces of spheres.
Microwell polystyrene plates coated with various proteins are commercially available. In particular, plates coated with streptravidin are used as tools in certain high throughput screening applications. For example, they are used to capture biotinylated antigens or antibodies. Applications in conventional ELISA use is also found in binding biotinylated oligonucleotides or PCR products in hybridization assays.
Streptavidin coated plates can be prepared by simply adsorbing the protein, in native form, onto the plate. The protein is commonly dissolved in a buffer, e.g., a carbonate/bicarbonate buffer, at a pH above 9.0 and then applied to the plates and dried. However, when the plates are so coated with native streptavidin only a limited number of protein molecules are bound or otherwise available to capture target molecules. In turn, the plates have a low capacity for capturing the intended target molecule. This results in diminished sensitivity of the assay and, accordingly, there is a need to increase the capacity of such coated plates and surfaces.
Now, in accordance with the present invention, there are provided new protein coated surfaces, which have a high capacity for capturing target molecules, thus yielding assays with enhanced sensitivity. Also, in connection therewith, assays using the coated surfaces provided by the present invention have an extended linear working range, particularly when the target molecules are small in size.
Surfaces prepared according to the present invention having the attributes identified above contain a coating consisting essentially of streptavidin, avidin or NeutrAvidin(trademark) deglycosylated avidin in polymeric form, wherein polymerization has been controlled to an extent such that the polymer is predominantly, i.e., greater than 50%, and generally at least 60%, dimers, trimers and tetramers of the native molecule. The extent of polymerization can be determined by SDS-PAGE electrophoresis. As used herein, the term xe2x80x9cconsisting essentially ofxe2x80x9d means that the coating contains the identified native molecules in polymeric form, but does not exclude the presence of a minor amount of native monomer.
In a preferred aspect of this invention, utilizing a coating buffer containing a lyotropic salt, such as potassium sulfate, at about neutral pH enhances coating of the polymerized protein onto a surface.